1. Field of the Invention
The invention relates to the substitution of an N-terminal amino acid in a peptide ligand of the thrombin receptor on platelets thereby conferring resistance to enzyme cleavage of the ligand and thus avoiding inactivation of platelet aggregating activity mediated by the ligand.
2. Description of the Related Art
Hemostasis is a complex process involving blood coagulation which proceeds along an extrinsic pathway or an intrinsic pathway. In both pathways, the final steps involve the formation of the enzyme thrombin which catalyzes the conversion of fibrinogen plasma protein into insoluble fibrin. In the intrinsic pathway, platelets adhere to a ruptured blood vessel and begin to clump. They then serve to accelerate the generation of the enzyme thrombin that causes blood to clot.
Thrombin also causes more platelets to clump and facilitates even more thrombin generation. Thrombin is a potent platelet agonist in vitro. It produces platelet shape change, aggregation and release of granule contents.
Thrombin's importance has stimulated research on the mechanism by which it activates platelets. A functional thrombin receptor has been identified on the surface membrane of platelets by Vu et al., Cell 64, 1057-1068 (1991).
Thrombin cleavage of the receptor produces a new amino terminal (SFLLRNPNDKYEPF, T-14) SEQ ID NO:1 tethered ligand peptide on the platelet surface. The peptide ligand is able to aggregate gel-filtered platelets directly. Therefore, thrombin induces platelet activation, at least in part, by cleaving the receptor on platelets, which permits the new amino terminus to function as a tethered ligand, interacting with another region of the receptor to induce activation signal(s).
Subsequent studies have shown that peptide ligands containing just the first five or six amino acids of T-14 are also active. (Vassallo et al. J. Biol. Chem. 267, 6081-6085 (1992); Hui et al., Biochem. Biophys. Res. Commun. 184, 790-796 (1992); Sabo et al., Biochem. Biophys. Res. Commun. 188, 604-610 (1992); Scarborough et al., J. Biol. Chem. 267, 13146-13149 (1992). Most groups found that the 6-mer is more active than the 14-mer.
The importance of a positive charge on the N-terminal serine was suggested from studies by the inventors herein, Coller et al., Biochem. 31, 11713-11720 (1992), and by others. Coller et al. and others also showed that acetylation of the amino terminus leads to loss of peptide activity and identified a correlation between peptide activity and the positive charge on the N-terminal serine. Coller et al., Id., and others (Vassallo et al., J. Biol. Chem. 267, 6081-6085 (1992); Scarborough et al., J. Biol. Chem. 267, 13146-13149 (1992)) showed that omitting the N-terminal serine of the ligand leads to loss of activity. The hydroxyl moiety of the N-terminal serine is not crucial for activity, however, since peptides in which alanine substitutes for serine retain substantial activity as shown by Sabo et al., Biochem. Biophys. Res. Commun. 188, 604-610 (1992); and Chao, et al., Biochem 31, 6175-6178 (1992).
Finally, Coller et al., Biochem. 31, 11713-11720 (1992) demonstrated that aminopeptidase M which is naturally occurring in plasma and serum and on endothelial cells, rapidly cleaves the N-terminal serine from peptide ligands, leading to loss of their ability to activate thrombin receptors. At micromolar concentrations of the peptide, the cleavage is rapid enough in platelet-rich plasma (PRP) to affect the platelet aggregation response. Even at millimolar concentrations, incubation with 50% plasma at 37.degree. C. leads to significant cleavage of the N-terminal serine by aminopeptidase M and loss of platelet aggregating activity.
Aminopeptidase M is the major enzyme responsible for the ability of plasma to cleave N-terminal neutral/basic amino acids from low molecular weight peptides (Ward et al., Biochem. Pharmacol. 40, 1725-1732 (1990); Ahmad and Ward, J. Pharmacol. Exp. Ther. 252, 643-650 (1990). Endothelial cells, smooth muscle cells, leukocytes, monocytes and other cells are known to contain aminopeptidase M (Look et al. J. Clin. Invest. 83, 1299-1307 (1989); Palmieri et al., Biochem. Pharm. 38, 173-180 (1989); Semenza, Rev. Cell. Biol. 2, 255-313 (1986)).
The rapid degradation and inactivation of the peptide ligand in the presence of plasma and serum makes it difficult to assess dose-response relationships precisely. In addition, an appreciation of the aminopeptidase cleavage and inactivation process raises the possibility that some of the observed effects of the peptides have resulted from only brief receptor activation, since dissociation of the peptide from the receptor may occur rapidly as the concentration of intact fluid-phase peptide decreases.
Although aminopeptidase M can be inhibited by compounds such as amastatin, leading to enhanced platelet aggregation responses of PRP to peptide ligand (Coller et al., Biochem. 31, 11713-11720 (1992)), amastatin and other aminopeptidase M inhibitors may have unknown effects on cells or plasma proteins. Acetylation of the N-terminus is the traditional method for producing a peptide that resists cleavage by aminopeptidase M, but acetylation of this ligand eliminates receptor activator activity.
Accordingly, it is an object of the invention to provide a means to prevent inactivation of the thrombin receptor peptide ligand.
It is a further object of the invention to provide a thrombin receptor peptide ligand that retains substantial functional activity but is resistant to cleavage by aminopeptidase M.
It is yet another object to provide a non-naturally occurring peptide that activates platelet aggregation.